CA1207252A - Vectors for cloning in streptomyces - Google Patents

Abstract

Abstract The present invention discloses selectable recombinant DNA cloning vectors for use in Strepto-myces.

Description

X-5512 -l-VECTORS FOR CLONING IN STREPTOMYCES
The present invention relates to novel re-combinant DNA cloning vectors comprising a functional origin of replication-containing restriction fragment of plasmid pN~100 and one or more DNA segments that confer resistance to antibiotics. The invention also relates to transformants of the aforementioned vectors.
Heretofore, the development and exploitation of recombinant DNA technology in Streptomyces and l related organisms has been retarded and made especially difficult because of the general lack of selectable genetic markers on cloning vectors. The vectors to which the present invention relates are functional and selectable in both Streptomyces and other host strains and therefore represent a significant advance in the technical art.
The vectors to which the invention relates are particularly useful because they are small, ver-satile, and can transform and be selected in any Streptomyces cell that is sensitive to an antibiotic for which xesistance is conveyed. Since over half ofthe clinically important antibiotics are produced by Streptomyces strains, it is desirable to develop cloning systems and vectors that are applicable to that industrially important group. The present invention ~7~

provides such vectors and thus allows for the cloning of genes into Streptomyces both for increasing the yields of known antibiotics as well as for the pro-duction of new antibiotics and antibiotic derivatives.
S The present invention provides vectors for cloning DNA into Streptomyces host cells and also allows for the convenient selection of transformants.
Since transformation is a very low frequency event, such a functional test is a practical necessity for determining which cell(s), of among the millions of cells, has acquired the plasmid DNA. This is important because DNA sequences that are non-selectable can be inserted onto the vectors and, upon transformation, cells containing the vector and the particular DNA
sequence of interest can be isolated by appropriate antibiotic selection.
For purposes of the present invention as disclosed and claimed herein, the follo~ling terms are as defined below.
Recombinant DNA Cloning Vector - any auto-nomously replicating agent, including but not limited to plasmids, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
Transformation - the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.

~72~2 X-56l2 ~3-Sensitive Host Cell - a host cell that cannot grow in the presence of a given antibiotic without a DNA segment that confers resistance thereto.
Insertional Isomer - one of the two or more possible recombinant DNA molecules formed when a DNA
fragment is inserted at one of two ox more compatible sites on the recipient DNA.
Plasmid pLR2 31. 6 kb B HI Restriction Frag-ment - essentially the same ~1.6 kb BamHI thiostrepton resistance-conferring fragment contained in plasmid pIJ6.
Plasmid pLRl or pLR4 ~3.4 kb BamHI Restric-tion Fragment - the same ~3.4 kb BamHI neomycin resis-tance-conferring fragment contained in plasmid pIJ2.
Amp - the ampicillin resistant phenotype.
TetS the tetracycline sensitive phenotype.
ThioR - the thiostrepton resistant phenotype.
Neo - the neomycin resistant phenotype.
The recombinant DNA cloning vectors to which the invention relates comprise:
a) a functional origin of replication-containing restriction fragment of plasmid pNM100 and b) one or more DNA segments that confer resis-tance to at least one antibiotic when trans-formed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.

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The vectors to which the invention relates are constructed by ligating a functional origin of replication~containing restriction fragment of plasmid pNM100 and one or more DNA segments that confer re-sistance to at least one antibiotic when transformedinto a sensitive host cell, said host cell being sus-ceptible to transformation, cell division, and culture.
Plasmid pNM100, from which origin of replica-tion-containing fragments are constructed, is ~9.1 kb and contains several restriction sites which are advantageous for molecular cloning. Since the origin of replication of plasmid pNM100 has been localized to within the ~3.8 kb BamHI restriction fragment, a variety of different origin ox replication-containing fragments can be generated by digesting the plasmid with restriction enzymes that cut outside the ~3.8 kb BamHI region. Plasmid pFJ143, an ~4 kb pNM100 deriva-tive from which additional pNM100 origin of replication-containing fragments are obtained, can also be used to construct the present invention. A detailed restric-tion site map of each of plasmids pNM100 and pFJ143 is presented in Figure 1 of the accompanying drawings.
to For purposes of the present application, Figure 1 and all subsequent figures are not drawn to scale.
P~asmid pNM100 can be conventionally isolated from Streptomyces virginiae/pNM100, a strain deposlted and made part of the permanent stock culture collection of the Northern Regional Research Laboratory, Peoria, Illinois, under the accession number NRRL 15156.
Plasmid pFJ143 can be conventionally isolated from Streptomyces ambofaciens/pFJ143, a strain similarly de-, 7ZS~

posited under the accession number ~REU. 15114. Both strains are available to the public as preferred sources and stock reservoirs of their respe~ti~e plasmids.
Although many di~erent orig.in o replica-tion~containins fragments of plasmid pN~100 cay be con-structed, those exemplified herein for illustrative purposes include the ~3.~ kb Bam~I restriction fragment of pNM100 and the ~4 kb BamHI restriction fragment of pFJ143. These ragments can be independently ligated to one or more antibiotic resistance-conferxi~g DNA
segments, exemplified herein for illustrative purposes by the thio~trepton resistance-conerring ~L.6 kb B~mHI
restriction fragment of plasmid pLR2, the neomycin resistance-conferring ~3;4 kb BamHI restriction frag-l ment of plasmid pLRl or plasmid pL~4 and the erythro~mycin resistance-conferring ~2.5 kb SalI-BamHI frasment of plasmid pIJ43 J to oxm vector illustrative of the present in~antion.
Plasmid pLR~, the source of the thiostrepton resistance-conferrins fragment, is ~18.~ kb and is constructed by ligating treated plasmid pIJ6, disclosed in Thompson et al., 1980, Nature 286:5~5, Jo HindIII-treated plasmid pBR322. Plasmid pLRl, the source of the neomycin resistance-conferring fragment,

2~ is ~14.8 kb and is similarly constructed except what plasmid pIJ2, disclosed in Thompson et al., 1980, is substituted for plasmid pIJ6. on analogous construc-tion, resulting in plasmid p~4, is made by ligating BamHI-treated plasmid pBR322 to BamHI-treated plasmid ~725Z

~5612 -6-pLRl. Plasmids pLR2, pLRl and pLR4 are functional in _. coli and therefore can be amplified and isolated conveniently for ~bsequent manipulat:ion.
Plasmid pIJ43, the source of the ~ry~hromycin resista~ce-conferring fragment, can be obtained from I.
coli 803/pIJ43, a strain deposited and made part of the permanent stock culture collection of the American Type Culture Collection, Rockville, Maryland. It is avail-able to the public as a preferred souxce and stocX
reservoir of the plasmid under the accession number ATCC 39156. A restriction site and functional map of each of plasmids pLRl, pLR2 end pLR4 is presented in Figure 2 of the accompanying drawings.
Por convenience and ease of construction, the thiostrepton resistance-con~erring ~1.6 kb B~mHI rag-ment, the neomycin resistance-conferring ~3.4 kb Bam~II
fragment and the erythromycin resistance-conf æ ring ~2.5 kb SalI-BamHI ragment are ligated to the ~3.8 k~
origin of replication-containing BamHI frasment of plasmid pN~100 or the ~4 kb origin of replication-containing BamHI fragment of plasmid p~Jl43. The resultlng recombinant D~A is then ligated to produce plasmids illustrative of the present invention Recombinant plasmids of two orientations result depend-ing upon toe orientation of the particular resistance-conferring PA fragment. Thus, ligation of the ~1.6 kb BamHI fragment of pla~mid pLR2 into the ~3.8 Xb BamHI
fragment of plasmid pNMlO0 results in illustrative plasmids pFJ204 an pFJ205; ligation of the 33.~ kb BamHI fragment of plasmid -LRl or plasmid pLR4 r2sults ~Z~-725;~

in illustrative plasmids pFJ206 and pFJ207; and liga-tion of both of the fragments results in illustrative plasmids pFJ208 and pFJ209. Similarly, ligation of the ~1.6 kb BamHI fragment into the ~4 kb BamHI fragment of plasmid pFJ143 results in illustrative plasmids pFJ170 and pFJ210; ligation of the 33.4 kb BamHI fragment results in illustrative plasmids pFJ211 and pFJ212;
ligation of both of the ~1.6 kb and 33.4 kb BamHI
fragments results in illustrative plasmids pFJ213 and pFJ214; and ligation of the ~2.5 kb SalI-BamHI fragment with an appropriate linker results in illustrative plasmids pFJ215 and pFJ216.
Various plasmid pNM100 restri.ction fragments can be used for ligat.ion of the antibiotic resistance-conferring DNA segments provided that the origin of replication contained in the ~3.8 kb BamHI restriction fragment is present. Such plasmid pNM100 restriction fragments include, but are not limited to, the ~9.1 kb BamHI, ~8.4 kb BamHI, ~4.7 kb BamHI, ~9.1 kb SacI, ~5.4 kb SmaI, and ~4.4 kb PvuII fragments. In addi-tion, a particular antibiotic resistance-conferring DNA
segment is not limited to a single position but can be ligated or inserted into varying sites of plasmid pNM100 or pFJ143 provided that the origin of repli-cation or other critical plasmid controlled physio-logical functions are not disrupted. Those skilled in the art undersiand or can readily determine which sites are advantageous for the ligation or insertion of a particular DNA segment.

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X-561~ -8 Although the thiostrepton, neomycin and erythromycin antibiotic resi~tance-conferring DNA
segments are respectively exemplified by the ~1.6 kb BamHI, ~3.4 kb BamHI, and ~205 kb SalI-~amH~ restric-tion fragments of pl3smids pLR2, pLRl and pIJ43, those skilled in the art can co~s~ruc~ and use, either individually or it combination, additional DN~ segments that also confer resistance to the aforementioned anti-blot sO Additional th~ostrepton resi~tance-conferring DN~ se~men~s of pla~mid ~L~2 include, for example, thy ~13 kb PstI restriction fragment and also the ~.8 B I subfragment of the kb ~amHI restriction fragment. Additional neomycin resistance-conferring DN~ sesment-~ of plasmid pLRl include, for example, the ~3.5 kb PstI restriction fragment and alto the larger l of the SstI-K~nI subfra~ments of the ~3.4 kb BamHI
restriction ra~ment. Additional fragment that conrer resistance to erythromycin include, for example the .8 kb SalI, ~2.7 kb SalI~ 3.0 kb H~-ndIII, ~2.8 kb XhoI~ II, and the ~4.1 kb EcoRI-BamHI restric-2~ tion fragments ox plasmid pIJ43.
Still other DNA segments that confer resis-tance Jo the same or to different antibiotics such as, for example, chloramphenicol, streptomycin, hygromycin, viomycin, tylosin and the like can also be constructed and used by those skilled in the axt. In addition, functional derivatives ox these or any ox the other antibiotic resistance-conferring DNA segments herein descried can be constructed by adding, eliminating, or substitutins certain nucleotides in accordance with the

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~-5612 _9_ genetic. code. Those skillet in the art will understand thaw lisation of these derivatives, or any other anti-hiotic resistance-conferring DNA segment Jo a pl smid pN~100 or pFJ143 origin of replication-containing fragment results in vectors that are also within the g scope of the present invention.
he restriction fragments of plasmids PN~lOO
and pF~143, and also tha various an~i~io~ic resistance-conferring DN~ segments, can be modified to facilitate ligation. For example, molecular linkers can be pro-vided to either or both of a par~ic~lar plasmid pNMlOOor pFJl43 restriction raqment or a particular resins-tance-conferring DNA segment. Thus, specific sites for qubsequent ligation can be co~struc~ed conveniently.
In addition, the origin of replication-containing l restriction fragments can also be modified by adding, eliminating, or substituting certain nucleotides to provide a variety o restriction sites for }igation of DNA. Those skilled in the art understand nucleotide chemis~y and the genetic code ana thus which nuc}eo-2~ tides are interchangeable and which DNA modificationsare desirablè for a specific purpose.
The present Stre~tomy~ functional vectors can also be ligated to a restriction frasment of an E.
coli plasmi~ such as, for example, pBR322~ pBR324, US pBR325, pBR328 and the like, to produce self replicating vectors that are selectable in both E. coli and Strep-tom~ces. These bifunctional constructions comprise the pWM100 origin of replication, a DNA segment that con-fers antibiotic resistance in Street , a replicon X-561~ -10-that is functional in E. coli and al50 a DNA segment that confers antibiotic resistance in ~:~ coli. Bi-functional constructions, exemplified herein by plas-mids pFJ219 and pFJ220, art parti~ular].y advantageous because amplification and manipulation of plasmids can be done Easter and more conveniently in E. coli than in ye. Thus, after desired recombinant D~A
procedures are accomplished within the I. coli host system, the entire plasmid or the particular mycPs DNA can be removed a re-constructed to plasmid ~0 foxm if necessary, end when transformed into a or related host cell.
The recombinant D~A cloning vectors of the present invention aye not limited for usa in a single specieS or-stxain of ye. To the contrary, the vectors are broadly applicable and can be trans-formed into host ceLls ox many Streptomy~s taxa, particularly restxictionless strains of economically Lmpor~ant taxa that produce antibiotics such as amino-glycoside, macrolide, ~-lactam, polyether and glyco-peptide antibiotics. Such restrictionless strains arexeadily selected and isolated from ye taxa by conventional procedures well known in the art (Lomovskaya et al , 1980, Microbiological Reviews 44:206). Yost cells of restrictionless strains lack restriction enzymes and thereore do not cut or degrade plasmid DNA upon transformation. For purposes of the prasent application, host cells containing restriction enzyme that do not cut any of the restxiction sites of the present vectors are also considered restriction-less.

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~-5612 Preferred host cells of restrictionless strainq of S~reptomyces taxa that produce aminoglyco-side antibiotics and iA which the present vectors are especially useful and can ke transformed, include restrictionless cells of, for example: S. kana-myceticus (kanamycins), S. chres~om~ceticus amino-sidine), S. (antibiotic PA 1267), S.
microseoreus ~anti~iotic SF-767), S, ribosidi~icus (antibiotic SF733), S. flavopersicus (spectinomycin), S actinospectacin)) S. rLmosus forma (paromomycins, catenuli~, S. fradiae vary italicus (aminosidine), S. bluensis var. bluensis (bluansomycin), S. catenulae (catenulin), S. olivo-reticul var. (destomycin A), S. tene-brarius (tobramycin, apr~mycin3, S. lavendulae (neo-13 mycin), S. albogriseolus (neomycins), S. albus ~ar.metamycinus (metamycin), S. ye æ~ var.
miensis ~spectinomycin), S. bikiniensis (streptomycin), S, 9~ (streptomycin)~ S- 5ll~gL ~3a a~al3Y~ vat-narutoensis (streptomycin), So poolensis (strepto-mycin), S. galbus (streptomycin), S. rameus (~tr~pto-mycin), S olivaceus (str~ptomycin), S. mashuensis (strèptomycin), S. hygrosco~ us var. limoneus (~alidam~-cins), S. rimofaciens (destomycins), S. hy~roscopicus fonma ~lebosus (glebomycin), S. fradiae (hybrimycins neomycins), S. eurocidicus (antibiotic A16316-C), S.
a~uacanus (N-methyl h~gxomycin B), S. cry~allinus (hygromycin I), S. noboritoe~sis (hysromycin), S.
hvgroscopicus (hygromycins), S. atrofaciens (hysro-7;~iZ

mycin), S. kasugaspinus (kasugamycins3, S.
I_ (kasugamycins), S antibiotic LL AM31), 5.
liYidus (li~idomycins~, S. hofuensis (se:Ldomycin com-__ .
plex), and S. canus (ri~osyl paromami~e),.
Preferred host cells of restrictionless trains f En t~xa that produce macrolide antibiotic and in which the present vectors are especially useful and can be transformed, include restrictionless cells of for example: S. caelestis (antibiotic M188), S. platensis (platenomycin), S.
rochei jar. ~olubilis ~anti~iotic ~2636), S. venezu~lae (methymycins), S. g___ eofuscus (bundlin), S. narbo-nensis ~josamycia, narbomycin), S. fun~icidicus (antibiotic NA-181), S. griseo~acien3 (antibiotic PA133A, B), S. roseocitreus ~albocycline), S. bruneo-lS ~albocycline), S. roseochromo~enes (albocycline), S ci~erochromo~Lenes (cineromycin B), S. albus (albo-_ mycetin), S. ~elleus ~argomycin, picromycin), S.
rochei (}ankacidin, borrelidin), S. ~iolaceoniger (lankaci~in)~ S. ~ris0us (borrelidin), 5. meizeus (ingramycin), S. albus var. ye (coleimycin), S. mycarofaciens ~acetyl-leukomyci~, espinomycin), S
y~roscopicus (turimycin, relomycin, maridomycin, tylosin, carbomycin), S. ~riseosp _alis ~relomycin), S.
lavendulae (aldgamycin), S. rimosus (neutramycin), S.
deltae (deltamycins), S. fun~icidicus vary espino-25myceticus (espinomycins), S. furdicidicus (mydeca-mycin), S. ambofac ens (foromacidin D), S. eurocidicus (methymycin), S. griseolus (griseomycin), S. flavo-chromogenes (a~aromycin, shincomycins), 5. fimbriatus 7~

~-5O12 -13-(amaromycin~, S. fasciculus (amaromycin), S. v (erythromycins), S. antibioticus (oleandomycin), S.
en (oleandomycin), _. spin jar. sura~aoensis ~kujimycins), S. kitasatoensis (leucomycin), S. narbonensis var. (leuco-mycin A3, josamycin~, S (miXonomycin)0 S. bikiniensis (chalcomycin), S. cirratus (cirxamycin), S. (niddamycin), S. v (angola-mycin), S. frldiae (tylosin~ lactenocin, macrocin), S.
~oshikiensis (bandamycin), S. griseofla w s (acumycin), 5. halstedil (carbomycin), S. tendae (carbomycin), S.
macrosporeus (carbomycin), S. thermotolerans (caxbo-. .
mycin), and S. albi iculi ~carbomycin)~
Preferred host cells of re~trictionles~st~ains of taxa what produce ~-lactam an.ibiotics and in which the present vectors are especially useul and can be transformed, include restric~ionless cell of, for example: S.
(A16~84, Lo 4550~ L~113902) S. (A16886B, clavulanic acid), S. lactamdurans (cephamycin C), S
griseus (cephamycin A, By, S. ~ygE~ æ~ (deacetoxy-cephalosporin O S. waday2mensis (WS-3442-D)~ S.
ch _ treusis (SF 1623), S. and S.
(C2081X); S. cinnamonensis, S. fimbriatus, S. h_lstedii, S. rochei and S. viridochromo~nes (cephamycins A, B);
US S. cattleYa (thienamycin); and S. olivaceus, S. flavo-virens, S. flavus, S. fuIvoviridis, S. ar~enteolus, and S sio~aensis (MM 4550 and MM 13902).
Preferred host cells of restrictionless stralns of Streptomyces taxa that produce polyather antibiotics and in which the present vectors are 3~

~Q~7252 ;~-561~ ~14-especially useful and can be transformed, include restric~io~less cells of, or example: I,. albus (A204, A~8695A and B, salinomycin), S. )icus (A218, emericid, DE3936), A120A, A28695A and I, etheromycin, dianemycin~, S. griseus (yrisorixin), S.
~ionomycin), S. eurocidicus var. 2ster dicus (laidlo-mycin~, S. lasaliensis tlaSalocid), S. ribosidLficus (lonomycin), S. cacaoi var. asoensis (lysocellin), S.
cinnamonensis (monensin), S. aureofaciens (narasin), S
gallinarius (RP 30504), So }ongwoodensis (lysocellin), S. flaveolus (CP38936), S. mutabilis (S-11743a), and S.
(ni~erici~).
Preerred host cells of restrictionless ~t~ains ox StreE~tom~ces taxa thaw produce glycopeptlde antlbiotics and in which the prevent vectors ore lS especially useful and can be transformed, include restric~ionless cells of, for example: S. orientalis and S. haranomachiensis (~ancomycin); S. candidus ~A-35512, avoparcin), and 5. (LL-A~ 374).
Preferred host cells of other yy~
restrictionless strains in which the presant vectors are especially useful and can by transformed, include restrictionless cells of, for example: S. coelic~lor, S. ~ranuloruber, S. roseos~orus, S. lividans, S.
enebraxius, S. espinosus, S. ac cins, S. , S. parvilin, S. pristinaesE_ralis, S. v~olaceoruber, S. vinaceus, S. vir~niae and S. azureus.
. I, In addition to the representative Stre~to-myces host cells described above, the present vectors are also useful and can be transformed into cells of res~rictionLess strains of other taxa such as, for ~2Q72S;~

~-5612 example: Bacillus, StaPhv-lococcus and related Actino-mycetes, includîng Streptosporan~ium, Ac , Nocardia, and Micromonospora. Thus, the vector of the present invention have wide application and are useful and can be transformed into host cells of a variety of organisms.
While all the embodiments of the pr sent invention are useful, some o the preset recombinant DNA cloning vectors and ~ransformants are preferred.
accordingly, preferred vectors are plasmid~ pN~100, pFJ204, pFJ207/ pFJ208, pFJ143, pFJ170, pFJ212, pFJ214, pFJ215 and pFJ220; and preferred tra~sormants are Stre~tomyce~ ambo~aciens/pN~100, S. amb ~aciens/pFJ~04, S. lividans/pFJ204, S. amboacien~pFJ~07, S. ambo-faciens!pFJ208, S. ambofaciens/pFJ143, S. ambofaciens~-l pFJ170, S. lividans/pFJ170, S. a~bofa iens/p~J212,S. ambofaciens/pFJ214, S. ambofaciens/pFJ215, S.
ambofaciensJpFJ220 and E. coli K12 HBlOl~pFJ220.
Moreover, of this preferred group, plasmids p~M100, pFJ143, pFJ170, pFJ204, pFJ207 and pFJ208 and trans-formants S. am~ofaciens/pN~I100, S. a~bofaciens/pFJ143,S. a~ofaciens/pFJ170, S. lividans/pFJ170, S. ambo-faciens/pFJ204, S. E~bofaciens/pFJ207 and S. ambo-~aciens~pFJ208 are most preerred.
The recombinant D~A cloning vectors and transform2nts of the present i~ention have broad utility and help ^ill the nerd for suitable cloning vehicles for use in Stre~tomyces and related organisms Moreover, the ab-lity of the present vectors to confer resistance to antibiotics that are toxic to non-trans~
fonmed host culls, also provides a functional means for 3~

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selecting transformants~ This is important because of the practical necessity for determining and selecting the pa~icular cells that have acquired Hector DN~
Additional DNA segments, that lack funct.ional tests or their presence, can also be inserted onto the present vectors and then transformants containing the non-selectable DN~ can by isolated by appropria! e anti biotic selection. Such non-selectable DNA segments can ye inserted at any site, except within regions neces-sary or plasmid function and xeplication, and include, l but are not limited to, genes ha specify antibiotic modification en~mes and regulatory gene3 of all types.
Morn particularly, a non-selectable DNA seg-ment that comprises a gene it inserted on a plasmid such as for example, illustrative plasmid pF~208, at US the central SalI restriction site of the kb Bam~I
resistance-conferring fragment. Such an insertion inactivates the thiostrepton resistance gene and thus allows for the easy identification of tra~sformants containing the recombinant plasmid. This is done by 2~ first selecting for neomycin resistance and, secondari-ly, identifying those neomycin resistant transormants that are not resistant to thiostr pton. In a similar manner, insertion o a DNA segment of interest at, for example, the internal BamHI restriction site of the ~3.4 kb BamHI resistance-conferring fragment inacti-vates the neomycin resistance gene. Thus, trans-fonmants carrying this recombinant plasmid also art identified easily by first selecting for thios~repton resistance and, secondarily, identifying those thio-3~

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X-5~12 -17-strepton resistant transformants what are not resistant to neomycin~ Similar selection invol~ins the insex-tional inactivation of the erythromycin glene can also be done. Therefore, ths anility to select for anti-biotic resistance ln Stre~tom~ce~ and related cells allows for the efficient isolation of the extremely rare cells that contain the particular non-selectable DNA of interest.
The functional test for antibiotic resis-tance, as described herein above, is also used to ~0 locate DNA segments that act as control elements and direct expression of an individual antibiotic resis-lane gene. Such segments, including buy not limited to, promoters, attenuators, repressors, inducers, ribosomal binding sites, and the like; are used to lS control the expression of other genes in cells ox Streptomyces and related organisms.
The thiostrepton, neomycin and erythromycin resistance-conferring vectors of the present invention are also useful for insuring that linked DNA segments 2~ are stably maintained in host cells over many genera-tions. These gene or DNA frasments, covalently linked to the thiostrepton, neomycin or erythromycin nets tance-con~erring fragment and propagated either in Stseptomyces or in the cells ox related orsanisms, are maintained by exposing the transorman~s to levels af thiostrepton, neomycin or erythromycin that are toxic to ~on-transSormed cells. Therefore, transror~ants that lose the vector, and conse~uen~ly any covalently lived DNA, cannot grow and are eliminated rrom the 3~

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culture Thus, the ve tors of the present invention can stabilize and maintain any DN~ sequence ox interest.
The clonlng vectors and transformants of the present invention proYide or the cloying o' genes to improve yields of various products that are currently 3 produced in ye and related cells Examples of such products include, but are jot limited to, Streptomycin, Tylo~in, Cephalo~porins, Actaplanin, Narasin, ~onensin, Apramycin, Tobramycin~ Erythromycin, Tetracycline, Chloramphenicol, and the like. The present invention also provides selectable vectors that are useful for cloning, characterizing and reconstruct-i~g DN~ sequences that code for commercially .important p~otainq such as, for example, human insulin, human proinsulin, glucaqon, interferon, human growth hormone, l bovine s~ow~h hormone and the like; or enzymatic functions in metaboLic pathways leading to commercially important processes and compaunds; or for control elements that improve gene expression. These desired DNA sequences include, but are not limited to, DNA what 2~ codes for enzymes that catalyze synthesis of deriva-tized an~ibio~ics such as, for example, St~eptomycin, Cephalosporin, ~ylosin, Ac~aplanin, Narasin, .~onensin, Apramycin, Tobramycin, Tetracycline, Chloramphenicol and ~rythromycin derivatives, or-~or enzymes that mediate and increase bioproduction of antibiotics or other products. The capability or inserting and stabiliæing such DNA segments thus allows for increas-i~g the yield and availability of antibiotics that are produced by Streptomyces and related organisms.

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~-;612 -19-Streptom~ces virq~niae/pNM100 and S. ambo-faci~ns/pFJ143, respective sources of plasmlds pNM100 and pFJ143, can be culturad in a number of ways using any ox several different media. Carbohydrate sources which are preferred in a culture medium in-clude, for example, molasses, glucose, dextrin and glycerol, and nitrogen sources include, for example, soy flour, amino acid mixtures and peptones. Nutrient inorganic salts are also incorporated and include the customary salts capable of vielding sodium, potassium, ammonia, calcium, phosphate, chloride, sulfate and like ions. us is necessary for thy growth and development o other microorganism, ~s3ential trace elements are also added. Such trace elements are commonly supplied as impurities incidental to the addition of other con-stituents of tha medium.
Stre~to~ces virginiae/pNM100 and S. ambo-faciens/pFJ143 are grown under aerobic culture condi-tions over a xelatively wide pH range of about S to 9 at temperatures ranging from about 15 to 40C. For production of plasmids pN~100 and pFJ143 in greatest quantity, however, it is desirable to start with a culture medium at a pi of about 7. 2 and maintain a culture temperature of about 30C. Culturing Stre~to-mvces virginiae/pNM100 and S. ambofaciens/pFJ143 underthe a'orementioned conditions results in a resarvoir of cells from which plasmids pN~100 and pFJ143 can be isolated my techniques well known in the art.
The hollowing examples further illustrate and detail the invention disclosed herein. Both an e~plana-tion of and the actual procedures for constructing the invention are described where appro?riate.

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X-561~ . -20-tsolation of Plasmid pNM100 . Culture of /pNM100 A vegetative inoculum of Stre~t~
_____/pNM100 (NR*L 15156) was conventionally prepared by growing the strain under submerged aerobic condition in 50 ml. of sterilized trypticase 50y broth* at 35 g./l. in deionized water 10 ' *-Trypticase soy broth.is obtained from Difco Laboratorie-~, Detroit, Michigan.
he trypticase 90y broth inoculum was incu-bated or 48 hours at a temperature of 30C. About ` lS 10 ml. of the incubated inoculum were first transferred to 500 ml. of sterilized broth and then incubated for about 20 hours at 30~C. The pi was not adjusted.
Ater incubation, the Streptomyces v~sLniae~p~100 cells were ready for harvest and subsequent isolation of plasmid DNA.
B. Plasmid Isolation About 10 g. (wet wgt) of Strep~omyces lr~niae~pNM100 cells were harvested by centrifugation (10 minutes, 5C., 10,000 rpm). The cells were homog-enized using a tissue grinder, washed in TES buffer (0.05M tris~hydroxymethyl)aminomethane [tris], 0.005M
EDTA, and 0.05M NaC1, pH 8~0), and then suspended in TES buCfer containing 25~ sucrosa. After the addition of about 120 mg. of lysozyme in 20 ml. of TES-25 X~5612 -21-sucrose buffex, the suspension was incubated at ~5037C.
fox about 20 minutes and, upon addition of 40 ml. uf 0.25M EDTA, pH 8.0, the suspension was again incubated at 35C. for 10 minutes. Following thi~3, about 40 ml.
ox 5~ SDS (sodium dodecyl sulfate) in TZ buffer tO.31M
iris O.OOlM EDTA, pH 8.0) was added and then, after the resultant mixture was again incubated it 3 -37~C.
for 20 minutes, a~ou~ 50 ml. of 5M NaCl in deionized water was added. The mixture was stirred, placed on an ice bath for about 4 hours and then centrifuged ~30 minutes, 4C., 10,000 rpm). About .313 volumes of 42%
polyethylene glycol in deionized water were added to the NaCl supernatant and the resulting mixture was cooled a 4C. for about 18 hour3. the DNA precipitate lS was collected by centrifugation US minute, 4C. r 3000 rpm) and was then dissolved in TES buffer at pH 8Ø
Centrifugation (40 hours, lSC., 35,000 rpm) using cesium chloride and ethidium chloride gradients sepa-rated the D~A into two well defined bands with the lower band constituting the desired plasmid p~M100. As an alternative, 4.14 g. cesi~m chloride can be dis-solved in 1.84 ml. of STE (10 mM Tris-HCl, pH 8, 10 mM
NaCl, 1 mM EDTA, p~8) and .5 ml. EDTA (.25~, pH 83.
About 1 ml. DNA suspension and .8 ml. ethidium bromide ~5 (5 mg./ml.) are added resulting in a 5.1 ml. gradient with 1.6 g./ml. cesium chloride and 800 ~g./ml. ethidium bromide. Centrifugation (5 hours, 20C., 60,000 r.p.m.3 in an ultracentrifuge with a vertical rotor, such as 3eckman VTi65, followed by deceleration for hours without breaking, results in well de~lned bands. The 25~
X~5612 ~22-lower band constitutPs the desired plasmld pNM100.
Following conventional procedure, the plasmid band was removed, washed twice with isoamyl alcohol, dialyzed over TE buffer at pi 8.0 and precipitated with ethanol.
The thus isolated plasmid pNM100 DNA was dissolved in .4 ml. of TE suffer at pH 8.0, and was then frozen at -20C~ for storageO
Example 2 Construction of Plasmid pLR2 A. ~ndIII Digestion of Plasmid pIJ6 .
About 20 20 ~g.) of plasmid pIJ~ DNA, disclosed in Thompson et al , 1980, Nature 286:525, 15 S l BSA~ovine Serum albumin, 1 mg./ml~), 19 l water, 1 l ox HindIII containing 3 New England Bio Labs units) restriction enzyme*, and 5 l reaction mix** were incubated at 37C. for 2 hours. The reac-tion was terminated by the addition of about 50 l of 20 4M ammonium acetate and 200 l o 95% ethanol. The resultant DN~ precipitate was washed twice in 70~
ethanol, dried in acuot suspended in 20 l o TE
huf~er, and frozen a -20C. for storage Restriction enzymes can be obtained from the follow-ing sources:
Jew England Bio Labs., Inc.
32 Tozer Road - 8e~erly, Massachusetts Ol91S
Boehringer-~annheim Biochemicals 7941 Castleway Drive Indianapolis, Indiana 46250 ~-5612 _~3 Bethesda Research Laboratories no P.O. Box 577 Çaithersburg, Maryland 20760 **
Reaction mix for ~indIII restriction enzyme was pre-pared with the hollowing composition.
600m~ NaCl lOOmM Tris-HCl, p~7.9 70mM MgCl~
1O~M Dithlothreitol B. HindIII Digestion of Plasmid pBP.322 bout 8 l (4 ~g.) of plasmid pBR322 DN~, 5 Ul. reaction six, 5 l BSA (1 mg./ml.), 31 l waxer, and 1 ill. o HindIlI res~iction enzyme were incubAted a 37C. or 2 hours. Aver the reaction was lS terminated by incubating at 60C. for 10 minutes, about 50 l of 4~1 ammoni~m acetate and 200 l of 95~
ethanol were added. The resultant DNA precipitate was washed twice in 70~ ethanol, dried in vacuo, and sus-pended in 4~ l of water.
C. Ligation of HindIII digested Piasmids pIJ6 ar.d , pfiR3~2 About 20 l of HindIII treated plasmid pIJ6 (prom example 2~, 20 l of ~indIII treated plasmid pBR322 tfrom Example 2B), S l BSA l mg./ml.), 1 l of T4 DNA ligase*, and 5 l ligation mix were incu-sated at 16C. for 4 hours. The reaction was ter-minated by the addition of about 50 l 4M ammonium acetate and 200 l of 95% ethanol The resultant DNA
precipitate was washed twice in 70% ethanol, dr,ed ln 72Si~

x-s 6 12 - 2 4 Yacuo and suspended in TE buffer. Toe suspended D~IA
constituted the desired plasmid pLR2.

*T4 ~NA ligase can be obtained from the! following source:
New England Bio Labs., Inc.
32 Tozer R~.
Beverly, Massachusetts 01915 ~*I.igat~o~ mix was prepared with the following composi-tion.
SOOmM Tris-~Cl, pH7.8 200mM Dithiothreitol l O OmM MyC12 1OmM ATP
Example 3 Co~str~ction ox E. coli K12 H~101/pLR2 About 10 ml. of frozen competent Eo coli K12 HB101 cells (Bolivar it al., 1977, Gene 2:75-93) were pelleted by centrifugation and then suspended in about ~0 10 ml. of 0.01~ sodium chloride. Next, the cells were pelleted again, resuspended in about 10 ml. of 0.03M
calcium chloride, incubated on ice for 20 minutes, pelleted a third time, and finally,.resuspended in 1.25 mlO or 0~03M calcium chloride. The resultant cell ~5 suspension was competent for subsequent transformation.
Plasmid pLR2 in TE buffer (prepared in Example 2C) was ethanol precipitated, suspended in 1;0 l of 30mM calcium chloride solution, and gently mixed in a test tube with about 200 lo of competent E.
coli K12 ~B101 cells. The resultant mixture was in-cubated on ice for about 45 minutes and then at 42C.

~7~5Z

~-5612 -2S-for about 1 minute. Next, about 3 ml. of L-broth (Ber~ani, 1951, J. ~ac~eriology 62:293) t:ontaining 50 ~g./ml~ of ampicillin was added. The mixture was incubated with shaking at 37C. or 1 hour and then played on L-agar (Miller, 1~72, Experiments in Molecu-lar Genetics, Cold Spring harbor Lab Cold Spring harbor, New York) containing ampicillin Surviving colonies were selected and jested for the expected phenotype (AmpR, TetS), and constituted the desired E.
coli K12 HB101/pLR2 trans,ormants.
Example 4 Construction of Plasmid pLRl Pla5mid pLRl wa9 prepared in substantial accordance with the teaching of Example 2~-C except what plasmid pI~2, disclosed in Thompson et al., 1980, Nature 286:525, was used in place of plasmid pI~6. The desired plasmid pLRl was suspended in TE buffer.
Example 5 Construction of E. coli R12 HB101/pLRl .. . .. . .. ..
The desired construction was carried out in substantial accordance with the teaching of Example 3 except that plasmid pLRl, rather than plasmid pLR2, was used for transformation. Surviving colonies were selected and tested for the e~Ypected phenotype (AmpR, Tet ), and constîtuted the desired E. coli K12 ~101/
pLRl transformants.

A. Partial BamHI Digestion of Plasmid pL~l . . .
S bout 10 l (10 ~g.) of plasmid pL~l, S l BSA
(1 mg./ml~), 29 l water, 1 l of ~amHI (diluted 1 4 with water restriction enzyme, and 5 tll. reaction mix*
were incubated at 37C. for lS mi~u~es. The reaction was termin~ed hy the addi~io~ of about SQ l of ammonium ac~ta~e and 200 l of 95% ethanol. The resultant DNA precipitate was washed twice in 70%
ethanol, dried in vacuo, and suspended in 20 water.

Reaction mix for BamHI restriction enzyme was pre pared with the hollowing composition.
l~SM NaCl H
60mM Tris-HCl, p 7.9 60mM MgC12 2~ B. BamHI Digestion of Plasmid pBR322 .... __ .
The desired digestion was carried out it sup-s~antial accordance with the teaching of Example 28 except that BamHI restriction ~n~yme was used in place o HindIII restriction enzyme. The digested plasmid 2S pBR322 was suspended in 29 l or water.
C. Ligation of Partial BamHI Digested Plasmid pLRl and Bam~I Digested Plasmid pBR32Z
The desired ligation was carried out in substantial accordance with the keaching of Example 2C. The resul-X-5612 -26a-tant ligated DNA was suspended in To buffer and constio tuted the desired plasmid pLR4.
Ex~p1e 7 Constxuction of E, coli ~12 HB101/p~R4 The desired construction was curried out in substantial accordance with the teaching of Example 3 except tAat plasmid pLR4, rather than plasmid pL~2, was used for transformation. Sur~iviny colonies were se-lected and tested for the expected phenotype (AmpR, TetS), and constituted the desired E. coli K12 ~101/
pLR4 transormants.

`` ~L;2~725;~

~-5612 ~27-~5 A. BamHI Digestion of Plasmid pLR2 and Isolation of the l 6 ~rePton ~sistan~e-Conferrin~

About 50 ~g. of plasmid pLR2 DNA, 10 l reaction mix, 10 I. BS~ (1 mg.~ml.), 29 l water, and 1 l (4 units/~l.) of Bam~I restriction enzyme are incubated at 37C. for 2 hours. After adding an equal l volume of 4~ ammonium acetate and 2.5 volumes of ~5%
ethanol, tha mixture it cooled at -20C. for about 18 hours to precipitate the DNA~ The DNA precipitate is collected by ce~tri~uga~ion and when suspended in about 50 l ox TE buff The desired ~1.6 kb B~mH~ re-lS striction frasment is isolated conventionally from theDNA suspension my asarose gel electrophoresIs in substantial accordance with the teaching of Davis, I. W. en al., 1980, A manual For Genetic Engineering, Advanoed Bacteriol Genetics, Cold Spring Harbor Labora-tories, Cold Spring Harbor, New York. Followingisolation, the fragment is resuspended in about 2a ul.
c TE buffer for subsequent ligation B. BamHI Digestion ox Plasmid pNM100 and Isolation 2S of the ~3.8 kb Orisin of Replication-containinq ragment The desired digestion and isolation are carried out in substantial accordance with the teaching of Examp'e 8A except that plasmid pN~100, rather than 3~

~2~'72~

X 5612 -2a-plasmid pLR2, is used. Following isolation, the ~3.8 kb fragment is suspended in about 50 l of TE suffer for subsequent ligationO
C. Litton About 1 ~g. of the ~3.8 kb Ba~HI fragment ox plasmid p~M100, 1 ~g. of the ~1~6 kb amHI restriction fragment of plasmid pL~2, 5 l ligation mix, 5 So (1 mg./ml.~, 25 l watex, and 5 l T4 DNA ligase (New England Bio Labs) are incubated at about 16 DC. or about 4 hours. After adding about 50 l of 4M ammonium acetate and about 30~ l of cold ethanol, the mixture is cooled to about -20C. for about 18 hours to pre-cipitate toe DNA. The DNA precipitate is collected by centrifugation, washed with 70% ethanol, collected again, and than suspended in 50 of medium P (Hop-wood and Wright 1978, J. Molecular and General Genetics 162:307) for subsequent transformation.
Recombinant plasmids of two orientations result because the ~1.6 kb BamHI resistance-conferring fragment can ye oriented in either direction. The resultant plasmids pFJ204 and pFJ205 can be transformed into appropriate host cells and then conventionally identified by restriction enzyme and agarose gel electrophoretic ana}ysis (Dais, I. W. e al 1980). A
restriction site and functional map of each of plasmids pF~204 and pFJ205 is presented in Figure 3 of the accompanying drawings.

7~:5~:

~-5612 -29-Example 9 Construction of ~3~ YE amboPaciens/pFJ204 and _ _,. , ."~_",. _ .
5. am~ofaciens/pFJ205 Using about l go of the DNA from Example 8C
and lXl08 protoplasts of streptomyces ambofaclens, a strain deposited and made part of the permanent stoc}c culture collection of the Northern Regional Research Laboratory, Peoria, Illinois, from which it is avail--able to the public under the accession number NRRL
2~20, the desired constructions are made in substantial accordance with the teaching ox International Put cation (of International Patent Application No.
PCT/GB79~000~5) No. WO79/OlL6g, Example I. The desired ~ransformants are.selected for thiostrepton resistance by overlaying the regenerating protoplasts with R2 medium (Hopwood and Wright, 1978, Molecular and General Genetics 162:30) top agar containing sufficient thio;
strepton to bring the final plate concentration to 50 ~s./ml. The resultant am ofaciens~pFJ204 and S. ambofaciens/pFJ205 thiostrepton resistant ___ coLonies are isolated according to known proeedures, cultured, and then conventionally identified by re-striction enzyme and agarose gel electrophoreticanalysis or the constitutive plasmids (Davis, R. W. et al., 1980). The transformant cultures are then used for subsequent production and isolation of their respec-tive plasmids.

X-~612 _30 -A Bam~I Digestion o Plasmid pLRl and Isolation of the ~3.4 kb Neom~cin ResistanceConfe:
The desired digestion and isolatisn are carried out in substantial accordance with the teaching of Example 8~. The ~3.4 kb ~amHI restriction ragment is suspended in about 20 l ox TE buffer for ~ubse-vent ligation.
B. Ligation Tbe ~3.4 kb Bam~I neomycin resistance-conferring restriction frasment i3 ligated Jo the ~3~8 kb Bam~I fragment of plasmid pNM100 (prapaxed in Example 8B) in substantial accordance with the teaching of Example 8C.
Recombinant plasmids of two orientations result because the ~3.4 kb BamHI resistance-conferring fragment can be oriented in either direction. The resultant plasmids pF~Q6 and pFJ207 cay be tran~orme~
into appropriate host cells and then conventionally identified by restriction enzyme and agarose gel electropho~etic analysis (Davis, R. WO et al., 1~80).
A restriction site and functional map of each of plas-mids pFJ206 and pF~207 is presented in Figure 3 of the accompanying drawings.

3~

~7~5~

~-561~ -31-Example 11 Construction of 5trep_omyces ambofacier.ls/pFJZ06 and S. bofl~c FJ211~ , Using about 1 ~g. of the DNA from Example 10 and lX108 protopla~ts of S~reptomyces ambofaciens (NRRL
No. 2420), the desired constructions are made in substantial accordance with the teaching of Inter-national Publication (of International Patent Appli-cation No. PCT/GB79/00095) No. W079/0116g, Example 2.
The desired transformants are selected for neomycin resistance by overlaying the regenerating protoplasts with R2 medium top agar containing suficient neomycin*
to bring the final plate concentration to 1 ~g./ml.
The resultant Lo ambofaciens/pFJ206 and S. ambofaciens/pFJ207 neomycin resistant colonies are isolated accordins to known procedures and then conventionally identified by restriction enzyme and electrophoretic analysis of the constitutive plasmids (Davis, R. W. _ al., 1980). The transformant cultures are then used for subsequent production and isolation of their respective plasmids.
_ Antibiotic neomycin can be obtained from Sigma, St.
Louis, Missouri.
Example 12 Construction of Plasmids ~FJ208 and ?~J209 Plasmid pFJ204, isolated from St~e~omvces ambofaciens/pFJ204 according to the procedure of Exam-ple 1, is partially digested with Bam~I restriction 25~

enzyme. The digestion is carried out by incubating about 20 ~g. of plasmid pFJ204 DNA, l l reaction mix, 10 l BS~ (1 mg./ml.), 39 l water, and 1 l of BamHI xestrictio~ enzyme prepared by diluting 2 l of enzyme in 8 l ox watex) at ambient temperature for about 15 minutes. After adding an equal volume of 4 ammoni~m acetate and 5 Yolumes ox 95% ethanol, the mixture is cooled at -20~C. for about 18 hours to precipitate the DNA. The DNA precipitate is collected by centrifugation, rinsed in 70% ethanol, dried in 10 vacuo J and when suspended it about 50 l of TE buffer.
.
The partial ~amHI digest is when ligated, in substantial accordance with the teaching of Example 8C, to the plasmid pL~l ~3.4 kb neomycin resistance-con~er-ring ~am~I fragment (prepared in Example lOA), to produce the desired plasmids. The insertionaL isomers of plasmids pFJ2~08 and pF~2~9 are also produced since plasmid pFJ204 has two BamHI restriction sites for the insertion of the neomyci~ resistance fragment. Reccm-binant plasmids of two orientations result because the 2~ ~3~4 kb Bam~I neomycin resistance-con~erring fragment can be oriented in either direction. The resultant plasmid~ pFJ208 and pFJ209 can be transfoxmed into appropriate host cells ana then conventionally identi-ied by restriction enzyme and agarose gel electro-phoretic analysis (Davis, R. W., et al., 1980). A
restriction site and functional map o each of plasmids pFJ208 and pF~209 is presented in figure 4 of the accompanying drawings.

3~

~7~

Construction of Lo ambofaciens/pFJ208 and S. a=~ofaciens/p~J209 Using 1 ~g. of the DNA from Example 12 and lX108 protoplasts of Streptom~ces amboraciens (~RRL No.
2420~, the desired constructions are made in substan-tial accordance with the teaching of International Publication (of International Patent Application No.
PCT/GB79/00095) No. W079/01169, Example 2. The desired transformants are selected rirst for ~hiostrepton resistance and then for neomycin resistance by the methods described in Examples 9 and lL above. The resultant ye ambofaciens~FJ208 and S.
ambofaclens/pFJ209 thiostrepton and neomycin resistant colonies are isolated according to known procedures and then conventionally identified by restrickion enzyme and electrophoretic analysis of the constitutive plas-mid (Davis, R. W. et al., 1980). The transformantcultu~es are then used ror subsequent production and iqolation of their respective plasmids~
Example 14 Isolation of Plasmid pFJ143 A. Culture of Streptomyces ambofaciens/pYJ143 , vegetative inoculum or Streptomvces ambo-facien~/pFJ143 ~RRL 15114) was conventionally prepared in substantial accordance with the teaching of Example lA.

~72~

B. is I3~l~c~r The desired isolation was carried out in substantial accordance with the teachia~g of Example 1 except thaw thy inoculum of Example 14~, rather than the inoculum of Example lA, was used. Thy isolated plasmid pFJ143 D~A was dissolved in 1 ml~ of 10 fold diluted TE buffer and was then frozen at -20C. for storage.
Example 15 10 Construction of Plasmids ~F3170 and pFJ210 A. Bam~I Digestion of Plasmid pFJL43 _ Thy desired digestion is carried out in substantial accordance with the teaching of Example 8A
except that plasmid pFJ143, rather than plasmid pLR2, is used. The DNA precipitate was collected by cen-trifugation, rinsed in 70~ ethanol, dried in vacuo, and then suspended in about 50 l of TE buffer.
B.
~5 About 1 lug. of the Bam~I digestion plasmid pFJ143 and 1 ~g. of the ~1.6 ~b E3amE~I restriction fragment ox plasmid pLR2 (prepared in Example 8A) were ligated in substantial accordance with the teaching of Example 8C. Recombinant plasmids of two orientations result because the ~1.6 kb BamHI resistance~conferrin~
fragment can be oriented in either direction. The resultant plas~ids pFJ170 and pFJ210 can be transformed into appropriate host cells and then conventionally identiried by restriction enzvme and agarose gel 72~5~

electrophoretic analysis (Davis, R. W. et al., 1980).
A restriction site and functional map of each 9f plasmids pFJ170 and pFJ210 is presented in Figure 5 of the ~cc~pa~ying drawings.
Example 16 Construction of Strep~omy~_s ambofac ns~pFJ170 and S. ambofacier.s/pFJ210 With the exception what DNA from Example 15C
is used, the desired constructions are made, co~v~n-l tionally identifiad and used for subsequent productionand isolation of plasmids pFJ170 and pFJ210 in sub-stantial accordance with the teaching of Example 9.
Example 17 construction of Pla~mid~ pF~211 an The desired constructions art made and con-ventionally identified in substantial accordance with the teaching of Example 10 except tot BamHI digested plasmid pFJ143, rather than the ~3.8 kb Bam~I fragment 2~ of pla~mid pNM100, is used. A restriction site and functional map of each of plasmids pFJ211 and pFJ212 is presented in Figure 5 of the accompanying drawings.
Example 18 Construction ox Streptomyces ambo~aciens~pFJ211 and 2~
S 9 ambofaciens/pFJ212 "
With the exception that DNA from Example 17 is used, the aesired constructions are made, ccnven-tionally identified and use for subsequent production 3~

~L2~

X-5612 -36- .

and isolation of plasmids pFJ2}1 and pFJ212 in 5U~-stantial accordance with the teachinq of Example 11.
I.
r_ The desired constructions are made and con-ventionally identified in substantial accordance with the teaching of Example 12 except that plasmia pFJ170, rather than plasmid pPJ204, is used The inse~tional isomers ox plaids p~J213 and pF~214 axe also produced l since plasmid pFJ170 has two ~amHI restriction sits for the insertion of the neomycin resistance ragmen ~ecombin~nt plasmids of two orientations result because the ~3.4 kb BamHI neomycin resistance-conferring frag-ment scan be oriented in either direction. A restric-tio~ size and functional map of each of plasmids pFJ213 and pFJ214 is presented in Figure 6 o the accompanying dra~ing3.
Example 20 Construction of Streptomyc~s a~bofaciens/pFJ213 and S. ambofaciens/pF~214 With the exception that DNA from Example 19 is used, the desired constructions are made, conven-tionally identified and used for subsequent production and isolation of plasmids pFJ213 and pFJ214 in sub-sta~tial accordance with the teaching ox Example 13 3~

5~

~-5612 37 Example 21 Construction of Plasmids pFJ21~ Lo Lo A. Culture of E. colt 803/pIJ43 and Isolation of -Plasmid ~J43 The desired culturing of E. coli 803/pIJ43 (ATCC 39156) and the subsequent isolation of plasmid pIJ43 are both carried out in substantial accoxdance with the teaching of Davis, R. W. et al., 19B0. The pIJ43 DNA is conventionally suspended in TE ~uf~er and then cooled to -20C. for storage.
B. Digestion and Isol tion of ~2.5 kb SalI-BamHI
Fragment of ~lasmid ~I~43 ..
15 bout 20 ~g. of plasmid pIJ~3 DNA, 10 l reaction mix*, 10 l BS~ (1 mg./ml.) 39 ater, and of SalI restriction enzyme prepared by diluting in such a manner that 1 l contains 60 New England Bio Labs Units) were incubated at ambient temperature for about 15 minutes. After addins an equal volume of 4M
ammonium acetate and 2 volumes of 95~ ethanol, the mixture was cooled at -20C. for about 18 hours to precipitate the D~A. Thy DNA precipitate was collected by centrifugation, rinsed in 70~ ethanol, dried ln vacuo, and then suspended in about Z0 l of TE buffer.
Following addition of about 5 l of ~amHI reaction mix, 5 l BSA (1 mg.~m.l.), 39 l watPr, and 1 l of BamHI restriction containing excess New England Bio Lab units), the mixture was incubated at 37C. for about 60 minutes. An eaual volume of lM ammonium 7~52 X-5612 -3a-acetate and 2 volumes of ~5~ ethanol were added and then the mixture was cooled at -20C. for bout l hours to precipitate the DNA. The DNA precipitate was collected by centrifugatio~. The desired ~2.5 kb SalI-Bam~I frasments are separated an.d isolated con-S ventionally by agarose gel eleckrophoresis (Davis, R. W. et al., 1980).

Reaction mix for SalI restriction enzyme was pre-pared with the hollowing compo~itio~.
1.5 M NaCl 60 mMTris-~Cl, pi 7.9 60 mM MgC12 60 mM 2-m~rcaptoethanol C. Addition of Bam~I Linkers Jo the ~2. S kb SalI-Bam~I Fragment ox Plasmid pIJ43 lS
The addition of BamHI linkers is carried out in substantial accordance with the teaching of Ullrich et al., 1977, Science 196:1313. The resultant fragment it treated with Bam~I restriction enzyme to produce the desired B~mHI sticky te~mini. Tha ~2.5 kb BamHI frag-ment is then isolated according to Xnown procedures and stowed or subsequent ligation.

BamHI[d(CG&ATCCG)] and other firers are readily US available at:
Collaborative Research Inc.
128 Spring Street Lexington, Massachusetts 02173 3~

~Z~72~

D. ~atlon About 1 ~g. of Bam~I digested plasmid pFJ143 (prepared in Example 15A) and 1 ~g. of. the ~2~5 kb fragment of plaid pIJ43 (prepaxed in Examples 21~ and I), are ligated in substantial accordance with the teaching of Example 8C. Recombinant plasmids of two orientations result because the ~2.5 k~ BamHI fragment can be oriented in either dixection. The resultant plasmids pFJ215 and pFJ216 can be transformed into appropriate host cells and then conventionally identi-10 fied by restriction enzyme and agarose gel electro-phoretic analyses (Davis, R. W. et al., 1980). A
restriction site and functional map of each of plasmid~
~FJ215 and pFJ216 i5 presented in Figure 6 of the accompanying drawings.
Example 22 Con~truc~ion ox Streptomyces ambofacien~/pFJ215 and .
S. ambofaciens/pFJ216 .
The desired constxuctions are made in sub-s~antial accordance with the teaching of Exampla 9 except that plasmid pFJ215 and pFJ216 DNA, rather than DNA from Example 8C, is used. The desired trans-formants are selected for erythromycin resistance by overlaying the regenerating protoplasts with R2 medium top agar containing surficient erythromycin to bring the plate concentration to iO ~g.~ml. The resultant Streptomyces ambofaciens/p~J215 and S. ambofaciens/-pFJ216 erythr~mycin resistant colonies are isolated according to known procedures, cultured, tested for 3~

7%5;~

thiostrepton resistance and then conventionally identi-fied by restriction enzyme and agarose gel eLectro-phoretic analysis of the constitutive plasmids. The desired transformants are then conventionally cultured for subsequent production and isolation of plasmids pFJ215 and pFJ216.
Example 23 Construction of Chimeric Plasmids pFJ219 and pFJ220 The desired chimeric plasmids are obtained by ligation of partial BamHI digest of plasmid pFJ170 (prepared according to the teaching of Example 19), and BamHI digested plasmid pBR322 (prepared in Example 6B), in substantial accordance with the ligation procedure lS ox Example 2. The desired chimeric plasmid DNA is collected by centri~ugation, washed with 70~ ethanol, dried ln vacuo, and then suspended in 50 l of TE
buffer. Recombinant plasmids of two orientations result because the restricted plasmid psR322 can be oriented in either direction. A restriction site and functional map of each of plasmids pFJ219 and pF~220 is presented in Figure 7 of the accompanying drawings.
Example 2~

25 Construction of E. coli K12 HB101/pFJ219 and E. coli K12 HB101/pFJ220 .
The desired constructions are made in sub-stantial accordance with the teaching of Example 3 ex-cept that plasmid D~A from Example 23, rather than plasmid pLR2, is used for the transformation. Surviv-X-5612 l ing colonles are first selected, tested for the ex-pected phenotype (~mpR, Tot end the conventionally identified as Rae desired E coli Kl~ ~B101/pF~219 and En old X12 ~B101/pFJ229 tra~sformants by restriction enzyme and agaxose gel electrophoretic analysis of the 5 constituti~e plasmids Dais R. W. et alg, 1980)~

Construction of e~Y~ ambofaciens/pFJ219 and S. ambofaciens/pFJ2~0 The ce-i~ed constructions are made in ~ub~
stantial accordance with the teaching of Example 9 ox cept that pla~mids pFJ219 nd pFJ22Q, rather than pla~mids p~J204 and pF3205, are used for the inns ormation. The resulting transformants are selected o~ thiostrepton resistance by the method described in Example 9 above . The thus constructed thios trepton resistant Str~to~es ambofaciens/pFJll9 and S.
amboacierss/pFJ220 colonies are isolated according to known procedures and then con~entio~ally identified by restriction enz~m~ and agarose gel electrophoretic analysis of the constitutiYe pl~smids.
Representative plaqmids and transfor~ants constructed in accordance with the foreyoing teaching include the following listed in Table 1 below.

~;~Q7~5~

,-i H h i-l I to H I (I C: h O I :1 O O O :~ O O
ul 1.l rl Ut O h :~ rl U3 Off t~J O ~1-l so O t:4 5 4 S O
H S a ~3 C4 H .S 0 æ
o u o o o 3 o 3 a o I: o o ~3 .c s O O rl l ~0 ta O H C4 tn l _I Us ~;4 W S H Y J
pa o Cl. 5 l Q~ :~1 $ ~b ~1 $
O IJ Off a e O m o m n o o rla) 0 OR on 0 o O e o a c o 1:: rl r ,1 Hi rl ~rl l 0 ~rl f rl ,~ 2: O So E us C) SDI 3 us l a so ~15i l C: O 'Y:1 I H C 4 H C
o w o a) if l if S l 1 W ,4 li a S: ~rl 41 E3 1 rl rl 41 En l ~rl l o ,~ o x l O O l l 0 o a) u om~: opt o al P
I rl rl .C O 1 C) O O on ago w w Jo .4 u3 (I l u rl So En la c O O aJ J
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Claims (59)

The embodiments of the invention for which an exclusive property or privilege is claimed are defined as follows:

1. A recombinant DNA cloning vector compris-ing:
a) a functional origin of replication-containing restriction fragment of plasmid pNM100 and b) one or more DNA segments that confer resis-tance to at least one antibiotic when trans-formed into a sensitive host cell, said host cell being susceptible to transformation, cell division, and culture.

2. The cloning vector of Claim 1 wherein the restriction fragment of pNM100 is the ~3.8 kb BamHI
restriction fragment.

3. The cloning vector of Claim 1 wherein the restriction fragment of pNM100 is the ~4 kb BamHI re-striction fragment of plasmid pFJ143.

4. The cloning vector of Claim 1 wherein one DNA segment confers antibiotic resistance to thio-strepton.

5. The cloning vector of Claim 1 wherein one DNA segment confers antibiotic resistance to neomycin.

6. The cloning vector of Claim 1 wherein one DNA segment confers antibiotic resistance to erythro-mycin.

7. The cloning vector of Claim 1 wherein one DNA segment is the ~1.6 kb BamHI restriction fragment of plasmid pLR2.

8. The cloning vector of Claim 1 wherein one DNA segment is the ~3.4 kb BamHI restriction fragment of plasmid pLRl.

9. The cloning vector of Claim 1 wherein one DNA segment is the ~2.8 kb SalI, ~2.7 kb SalI-BglII, ~3.0 kb HindIII, ~2.5 kb SalI-BamHI, ~2.8 kb XhoI-BglII, or the ~4.1 kb EcoRI-BamHI restriction frag-ment of plasmid pIJ43.

10. The recombinant DNA cloning vector of Claim 1 which is plasmid pFJ204, pFJ205, pFJ206, pFJ207, pFJ208, pFJ209t pFJ170, pFJ210, pFJ211, pFJ212, pFJ213, pFJ214, pFJ215, pFJ216, pFJ221, pFJ222, pFJ223, pFJ224, pFJ225, pFJ226, pFJ227, pFJ228, pFJ229, pFJ230, pFJ231, pFJ232, pFJ233, pFJ234, pFJ235, pFJ236, pFJ237, pFJ238, pNM101, pNM102, pNM103, pNM104, pFJ265 or pFJ266.

11. The cloning vector of Claim 10 which is pFJ204.

12. The cloning vector of Claim 10 which is pFJ205.

13. The cloning vector of Claim 10 which is pFJ206.

14. The cloning vector of Claim 10 which is pFJ207.

15. The cloning vector of Claim 10 which is pFJ208.

16. The cloning vector of Claim 10 which is pFJ209.

17. The cloning vector of Claim 10 which is pFJ170.

18. The cloning vector of Claim 10 which is pFJ210.

19. The cloning vector of Claim 10 which is pNM101.

20. The cloning vector of Claim 10 which is pFJ212.

21. The cloning vector of Claim 10 which is pFJ213.

22. The cloning vector of Claim 10 which is pNM103.

23. The cloning vector of Claim 10 which is pFJ215.

24. The cloning vector of Claim 10 which is pFJ216.

25. The cloning vector of Claim 10 which is pFJ222.

26. The cloning vector of Claim 10 which is pFJ224.

27. The cloning vector of Claim 10 which is pFJ233.

28. The cloning vector of Claim 10 which is pFJ235.

29. The cloning vector of Claim 10 which is pFJ265.

30. The cloning vector of Claim 10 which is pFJ266.

31. Plasmid pNM100.

32. Plasmid pFJ143.

33. The ~3.8 kb BamHI restriction fragment of plasmid pNM100.

34. The ~4 kb BamHI restriction fragment of plasmid pFJ143.

35. A recombinant DNA cloning vector which comprises a replicon that is functional in E. coli, a DNA segment that confers antibiotic resistance in E.
coli and a restriction fragment comprising a plasmid of Claim 1.

36. The cloning vector of Claim 35 in which the replicon that is functional in E. coli and the DNA
segment that confers antibiotic resistance in E. coli comprise a restriction fragment of plasmid pBR322, pBR324, pBR325, or pBR328.

37. The cloning vector of Claim 30 which is plasmid pFJ219, pFJ220, pFJ239, pFJ240, pFJ241, pFJ242, pFJ243, pFJ244, pFJ245, pFJ246, pFJ247, pFJ248, pFJ249, pFJ250, pFJ252, or pFJ253.

38. The cloning vector of Claim 37 which is pFJ219.

39. The cloning vector of Claim 37 which is pFJ239.

40. The cloning vector of Claim 37 which is pFJ247.

41. The cloning vector of Claim 37 which is pFJ253.

42. A transformed host cell comprising a recombinant DNA cloning vector of Claim 1.

43. A transformed host cell comprising a recombinant DNA cloning vector of Claim 10.

44. The host cell of Claim 43 which is Streptomyces.

45. The host cell of Claim 44 which is Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces griseofuscus, Streptomyces fradiae, Streptomyces lividans, Streptomyces granuloruber, Streptomyces tenebrarius, or Streptomyces cinnamonensis.

46. The host cell of Claim 45 which is Streptomyces ambofaciens/pFJ204.

47. The host cell of Claim 45 which is Streptomyces griseofuscus/pFJ205.

48. The host cell of Claim 45 which is Streptomyces aureofaciens/ pFJ206.

49. The host cell of Claim 45 which is Streptomyces fradiae/pFJ207.

50. The host cell of Claim 45 which is Streptomyces ambofaciens/pFJ208.

51. The host cell of Claim 45 which is Streptomyces ambofaciens/pFJl70.

52. The host cell of Claim 45 which is Streptomyces ambofaciens/pFJ265.

53. The host cell of Claim 45 which is Streptomyces ambofaciens/pFJ266.

54. A transformed host cell comprising a recombinant DNA cloning vector of Claim 35.

55. A transformed host cell comprising a recombinant DNA cloning vector of Claim 36.

56. The host cell of Claim 55 which is E.
coli comprising a cloning vector of Claim 37.

57. The transformed host cell of Claim 55 which is E. coli K12 HB101/pFJ219.

58. The transformed host cell of Claim 55 which is E. coli K12 HB101/pFJ239.

59. The transformed host cell of Claim 55 which is E. coli X12 HB101/pFJ253.